The purpose of this study is to determine the genetic basis of Fanconi anemia (FA), an autosomal recessive disorder characterized by diverse congenital abnormalities, and a predisposition to bone marrow failure and malignancy, particularly acute myelogenous leukemia (AML). The specific aims of this project are: (1) to isolate and clone FA genes by functional cloning; (2) to determine the chromosomal location of these genes and to study their structure and expression; (3) to detect mutations in FA genes in DNA from affected individuals and to make genotype-phenotype correlations; (4) to develop screening methods using DNA technology for FA diagnosis and carrier detection. A major resource and unique feature of this proposal is our access to a large number of patients with FA exhibiting the full spectrum of its diverse features, through the International Fanconi Anemia Registry (IFAR) maintained by us at the Rockefeller University. This provides us with phenotypic information on FA patients as well as a source of cells for molecular studies. Understanding the genetic defect in FA should lead to a better understanding of birth defects and cancer predisposition in general, and the interaction of genetic and epigenetic factors in their pathogenesis. We are currently applying specific aims 2-4 to the study of the Fanconi anemia complementation group C (FACC) gene, the only FA gene isolated to date. Lymphoblastoid cell lines which have been characterized as non-C, and which are suitable for isolating genes for other FA complementation groups by functional cloning, have been developed in our laboratory. Hypersensitivity of the FA cells to DNA crosslinking agents will be used as a selectable marker; a high quality cDNA library in extrachromosomally replicating (EBNA)-based mammalian expression vector p-lambdaDR2 will be used for transfections. Once we have isolated the normal cDNA copy of an FA gene, we will determine its nucleotide sequence and perform a computer analysis to determine the extent of homology to other genes. Gene structure will be analyzed by exon mapping and transcription initiation sites will be studied. Gene expression will be studied by Northern blotting. The chromosomal location of cloned genes will be determined by in situ hybridization. DNA from FA patients will be screened for mutations using single-strand conformation polymorphism (SSCP) analysis; PCR- amplified fragments exhibiting mutations will be sequenced. Amplification refractory mutation system (ARMS) assays will be developed for FA diagnosis and carrier detection, the complementation group of individual patients will be determined by mutation detection. Linkage studies will be used to estimate the relative frequency of different complementation groups in the population.